Transcriptomic characterization of the enzymatic antioxidants FeSOD, MnSOD, APX and KatG in the dinoflagellate genus Symbiodinium

Background The diversity of the symbiotic dinoflagellate Symbiodinium sp., as assessed by genetic markers, is well established. To what extent this diversity is reflected on the amino acid level of functional genes such as enzymatic antioxidants that play an important role in thermal stress tolerance of the coral-Symbiodinium symbiosis is, however, unknown. Here we present a predicted structural analysis and phylogenetic characterization of the enzymatic antioxidant repertoire of the genus Symbiodinium. We also report gene expression and enzymatic activity under short-term thermal stress in Symbiodinium of the B1 genotype. Results Based on eight different ITS2 types, covering six clades, multiple protein isoforms for three of the four investigated antioxidants (ascorbate peroxidase [APX], catalase peroxidase [KatG], manganese superoxide dismutase [MnSOD]) are present in the genus Symbiodinium. Amino acid sequences of both SOD metalloforms (Fe/Mn), as well as KatG, exhibited a number of prokaryotic characteristics that were also supported by the protein phylogeny. In contrast to the bacterial form, KatG in Symbiodinium is characterized by extended functionally important loops and a shortened C-terminal domain. Intercladal sequence variations were found to be much higher in both peroxidases, compared to SODs. For APX, these variable residues involve binding sites for substrates and cofactors, and might therefore differentially affect the catalytic properties of this enzyme between clades. While expression of antioxidant genes was successfully measured in Symbiodinium B1, it was not possible to assess the link between gene expression and protein activity due to high variability in expression between replicates, and little response in their enzymatic activity over the three-day experimental period. Conclusions The genus Symbiodinium has a diverse enzymatic antioxidant repertoire that has similarities to prokaryotes, potentially as a result of horizontal gene transfer or events of secondary endosymbiosis. Different degrees of sequence evolution between SODs and peroxidases might be the result of potential selective pressure on the conserved molecular function of SODs as the first line of defence. In contrast, genetic redundancy of hydrogen peroxide scavenging enzymes might permit the observed variations in peroxidase sequences. Our data and successful measurement of antioxidant gene expression in Symbiodinium will serve as basis for further studies of coral health.

Symbiont diversity and coral bleaching: An antioxidant view on thermal stress

Thomas Krüger

The functioning of coral reef systems, as biodiversity hotspots, is largely dependent on the symbiotic association between dinoflagellate symbionts (Symbiodinium spp.) and scleractinian coral hosts. The breakdown of this symbiosis (coral bleaching), as a result of global warming and other stressors, therefore has profound implications for the tropical marine environment. Corals associate with a variety of Symbiodinium genotypes, and it is this mosaic nature that contributes to the variable stress thresholds of corals. Research over the past 25 years has established that the generation and scavenging of reactive oxygen species (ROS) in both partners, under light and thermal stress, is a fundamental element of the bleaching response. However, while the existence of more thermally susceptible and tolerant symbiont types has been recognized, the differences in the antioxidant systems that may accompany these properties have received less attention. The purpose of this thesis was to explore the role of the antioxidant network in explaining the different thermal susceptibilities of various symbiont types and how the activity of key antioxidants in both partners under thermal stress relates to bleaching patterns in different corals. Thus, the specific objectives were to: (1) assess the antioxidant network response in different Symbiodinium types; (2) investigate the activity and structural diversity of key enzymatic antioxidants in different Symbiodinium types; (3) examine the regulation of these antioxidants at the transcriptomic and proteomic levels; and (4) contrast the symbiont’s and host’s antioxidant responses under bleaching conditions. Symbiodinium types in culture were found to differ significantly with regards to the concentration and activity of specific antioxidants, exhibiting magnitude scale differences in some of them. However, the response of the main removal pathway, involving superoxide dismutase (SOD) and ascorbate peroxidase (APX), under lethal thermal stress was fairly similar. Instead, the typespecific differences were found to lie in more downstream systems, and particularly in those associated with the maintenance of the glutathione redox state. A declining glutathione redox state was the common feature of the three thermally susceptible Symbiodinium types: B1, C1, and E. Indeed, in comparison to the most sensitive type (B1), the tolerant type F1 exhibited stronger antioxidant up-regulation and the successful preservation of the highly reduced glutathione pool. Comparing antioxidant gene orthologues from members of different Symbiodinium clades (A-E) revealed a higher degree of sequence variation at the amino acid level for peroxidases, which reflected the genetic radiation of the genus. In contrast, primary defences in the form of SOD isoforms were highly conserved. Sequence variations between Symbiodinium types involved residues that constitute binding sites of substrates and co-factors, and therefore likely affect the catalytic properties of these enzymes. While expression of antioxidant genes was successfully measured in Symbiodinium B1, it was not possible to assess the link between transcriptomic expression and proteomic activity due to high variability in expression between replicates, and little response in their enzymatic activity over three days. In contrast to previous findings, up-regulation of antioxidant defences was not evident in Symbiodinium cells inside the host (i.e. in hospite). In fact, oxidative stress in the thermally sensitive corals Acropora millepora and Pocillopora damicornis was only apparent from increased host catalase activity, which interestingly preceded photosynthetic dysfunction of their symbionts. Baseline antioxidant activities of thermally tolerant and susceptible host species showed no differences, though the scavenging activities of the hosts were considerably higher than those of the symbionts. Baseline activities for the symbionts were different, however, with Symbiodinium C15 from the thermally tolerant coral Montipora digitata exhibiting the lowest activities for SOD and catalase peroxidase. This thesis provides significant findings with respect to the variability in antioxidant activity, structure, and network response in different Symbiodinium types in culture, and how these relate to thermal tolerance. What effect these differences have on the response in the intact symbiosis remains unclear, however, as the findings contradict the classic bleaching model of photoinhibition and symbiont-derived ROS. I argue, using previously published data, that heating rates might profoundly affect the way we perceive the antioxidant response of both partners to thermal stress, and that host antioxidant defences might not be as easily overwhelmed by symbiont ROS as suggested previously. This thesis reports important findings on the antioxidant system in different Symbiodinium types, but also raises new questions about the antioxidant response of the intact coral.

Victoria University of Wellington2014

Differential coral bleaching—Contrasting the activity and response of enzymatic antioxidants in symbiotic partners under thermal stress

Thomas Krüger

Mass coral bleaching due to thermal stress represents a major threat to the integrity and functioning of coral reefs. Thermal thresholds vary, however, between corals, partly as a result of the specific type of endosymbiotic dinoflagellate (Symbiodinium sp.) they harbour. The production of reactive oxygen species (ROS) in corals under thermal and light stress has been recognised as one mechanism that can lead to cellular damage and the loss of their symbiont population (Oxidative Theory of Coral Bleaching). Here, we compared the response of symbiont and host enzymatic antioxidants in the coral species Acropora millepora and Montipora digitata at 28 °C and 33 °C. A. millepora at 33 °C showed a decrease in photochemical efficiency of photosystem II (PSII) and increase in maximum midday excitation pressure on PSII, with subsequent bleaching (declining photosynthetic pigment and symbiont density). M. digitata exhibited no bleaching response and photochemical changes in its symbionts were minor. The symbiont antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and catalase peroxidase showed no significant upregulation to elevated temperatures in either coral, while only catalase was significantly elevated in both coral hosts at 33 °C. Increased host catalase activity in the susceptible coral after 5 days at 33 °C was independent of antioxidant responses in the symbiont and preceded significant declines in PSII photochemical efficiencies. This finding suggests a potential decoupling of host redox mechanisms from symbiont photophysiology and raises questions about the importance of symbiont-derived ROS in initiating coral bleaching.

Elsevier2015

Antioxidant plasticity and thermal sensitivity in four types of Symbiodinium sp.

Thomas Krüger

Warmer than average summer sea surface temperature is one of the main drivers for coraltemperature is one of the main drivers for coral bleaching, which describes the loss of endosymbiotic dinoflagellates (genus: Symbiodinium) in reef-building corals. Past research has established that oxidative stress in the symbiont plays an important part in the bleaching cascade. Corals hosting different genotypes of Symbiodinium may have varying thermal bleaching thresholds, but changes in the symbiont's antioxidant system that may accompany these differences have received less attention. This study shows that constitutive activity and up-regulation of different parts of the antioxidant network under thermal stress differs between four Symbiodinium types in culture and that thermal susceptibility can be linked to glutathione redox homeostasis. In Symbiodinium B1, C1 and E, declining maximum quantum yield of PSII (F-v/F-m) and death at 33 degrees C were generally associated with elevated superoxide dismutase (SOD) activity and a more oxidized glutathione pool. Symbiodinium F1 exhibited no decline in F-v/F-m or growth, but showed proportionally larger increases in ascorbate peroxidase (APX) activity and glutathione content (GSx), while maintaining GSx in a reduced state. Depressed growth in Symbiodinium B1 at a sublethal temperature of 29 degrees C was associated with transiently increased APX activity and glutathione pool size, and an overall increase in glutathione reductase (GR) activity. The collapse of GR activity at 33 degrees C, together with increased SOD, APX and glutathione S-transferase activity, contributed to a strong oxidation of the glutathione pool with subsequent death. Integrating responses of multiple components of the antioxidant network highlights the importance of antioxidant plasticity in explaining type-specific temperature responses in Symbiodinium.

Wiley-Blackwell2014

Antioxidant plasticity and thermal sensitivity in four types of Symbiodinium sp.

Thomas Krüger

Wiley-Blackwell2014

Symbiont diversity and coral bleaching: An antioxidant view on thermal stress

Thomas Krüger

Victoria University of Wellington2014